JP7360752B2 - Severe fever thrombocytopenia syndrome virus gene detection composition and method for diagnosing severe fever thrombocytopenia syndrome using the same - Google Patents

Description

The present invention relates to a composition for detecting a virus gene of severe fever and thrombocytopenia syndrome and a method for diagnosing severe fever and thrombocytopenia syndrome using the same.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute febrile disease caused by the SFTS virus, and is characterized by symptoms such as decreased white blood cell and platelet values, increased liver function values, and proteinuria. A tick infected with the SFTS virus bites a person and inhales the tissue fluid, causing infection, and the vectors are Haemaphysalis longicornis, Amblyomma testudinarium, and Ixodes nipponensis. ) etc.

The incidence of SFTS patients is increasing nationwide in South Korea, and the mortality rate decreased from 2013 to 2016 by 47.6%, 29.1%, 26.6%, and 11.5% due to increased awareness of SFTS. Although this was a trend, the number of deaths rapidly increased in 2017 to 22.3%, an increase of 10.8% from the previous year. Therefore, since the number of SFTS patients is expected to continue to increase due to the increase in the tick population due to global warming, rapid and accurate diagnosis of SFTS infection is essential for improving patient prognosis.

Currently, severe fever thrombocytopenia syndrome is diagnosed by using the patient's blood to isolate the virus, by using the patient's serum to confirm an increase in antibody titer by IFA or ELISA analysis, or by using the patient's serum or plasma. There is a method of testing by amplifying SFTS virus RNA in the patient's blood using real time reverse transcription polymerase chain reaction (real time RT-PCR) or nested RT-PCR.

Among these methods, the method of diagnosing by culturing the virus requires a BSL3 facility, and culture may take some time, making it difficult to carry out in general hospitals.The test method mainly used for diagnosis is serum These are the indirect immunofluorescence antibody method and the immunoenzyme method, which are scientific tests. However, these methods are also prone to false-positive reactions due to cross-reaction with other pathogens, and it takes several days from the onset of symptoms to the formation of antibodies. The disadvantage is that follow-up tests are required for diagnosis.

In particular, the PCR method for detecting virus-specific genes from patient's blood in order to quickly diagnose SFTS virus at the early stage of the disease includes the traditional method of conventional RT-PCR and a modified version of this method called conventional RT-PCR. There are nested RT-PCR methods that are 100 times more sensitive than conventional methods, and real-time RT-PCR methods that allow you to check the results in real time while PCR is in progress. Severe fever thrombocytopenia syndrome virus is a single-stranded RNA virus that belongs to the genus Bunyaviridae and phlebovirus, and the virus genome is composed of Large (L), Medium (M), and Small (S) segments. . Real-time RT-PCR and nested RT-PCR are mainly used to target these three segments (L, M, and S segments) as RT-PCR diagnostic methods for detecting SFTS viruses, but nested RT - In the case of PCR, sensitivity increases by performing PCR twice, but performing PCR twice takes more manpower and time than necessary, and the possibility of potential contamination is higher, so it is difficult to increase sensitivity even with just one PCR. The reality is that the development of new, superior diagnostic methods is extremely urgent.

On the other hand, the three segments of the SFTS virus are the L segment that encodes RNA-dependent RNA polymerase and RdRp, the M segment that encodes glycoprotein (Gn and Gc), which are components of the viral membrane protein, and the viral nucleoprotein derived from the SFTS virus. Among these proteins, the S segment encodes nucleocapsid protein (NP), which is highly expressed and induces a cellular immune response in the infected host, and nonstructural protein (NSs), which has an immune evasion function. SFTS is mainly endemic in South Korea, China, and Japan, and when performing phylogenetic analysis based on the base sequences of the M and S segments of the SFTS virus, it was found that the genetic sequences of the viruses that occur in each country are different. , Chinese (C) clade, and Korean (K) clade.

Therefore, in order to quickly and accurately diagnose and treat SFTS infection, we need a sensitivity that allows us to quickly diagnose all the SFTS virus genes of the Japanese (J) clade, Chinese (c) clade, and Korean (K) clade at the early stage of the disease. What is most important is the development of diagnostic methods with excellent specificity.

Therefore, in consideration of these points, the present invention aims to provide a composition for detecting the SFTS virus gene, thereby providing a method for confirming the SFTS virus and diagnosing severe fever thrombocytopenia syndrome.

Another object of the present invention is to provide a kit for diagnosing severe fever thrombocytopenia syndrome (SFTS), which includes the composition for detecting the SFTS virus gene of the present invention.

Another object of the present invention is to provide a method for providing information for diagnosing Severe Fever Thrombocytopenia Syndrome (SFTS) infection.

In order to achieve the above objects, the present invention provides a method for treating Severe Fever with Thrombocytopenia Syndrome (SFTS), which comprises a pair of primers of SEQ ID NO: 2 and SEQ ID NO: 3; and a probe of SEQ ID NO: 4; A composition for detecting a virus gene is provided.

In one embodiment of the present invention, a pair of primers of SEQ ID NO: 2 and SEQ ID NO: 3; and a probe of SEQ ID NO: 4; It may be one that specifically binds to the S segment (S segment) of the Thrombocytopenia Syndrome virus.

The present invention also provides a kit for diagnosing severe fever thrombocytopenia syndrome (SFTS), which includes the composition for detecting the SFTS virus gene of the present invention.

The present invention also provides the following steps: (1) isolating viral RNA from a sample isolated from a patient suspected of having severe fever and thrombocytopenia syndrome; (2) synthesizing cDNA from the RNA; (3) as described above. Performing real-time RT-PCR amplification using the kit of claim 3 using the cDNA synthesized in step (2) as a template; and (4) confirming the gene of severe fever thrombocytopenia syndrome virus through step (3) above. Provided is a method for providing information for diagnosing Severe Fever Thrombocytopenia Syndrome (SFTS) infection, including the steps of:

In one embodiment of the present invention, the sample in step (1) above may be blood, tissue, cells, serum, plasma, saliva, sputum, or urine obtained from a mammal; or a tick;

In one embodiment of the present invention, synthesizing cDNA from the RNA in step (2) involves performing a reverse transcription reaction from the RNA genome of the SFTS virus through the action of reverse transcriptase. Alternatively, complementary DNA (cDNA) may be synthesized separately.

In one embodiment of the present invention, the confirmation of the severe fever thrombocytopenia syndrome virus gene in step (4) is performed using a method selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, and phosphorescence measurement. It may be accomplished by one or more of the following methods.

In one embodiment of the present invention, the gene of the severe fever thrombocytopenia syndrome virus in step (4) may be the S segment (S segment) of the severe fever thrombocytopenia syndrome virus consisting of SEQ ID NO: 1. .

The composition for detecting the severe fever thrombocytopenia syndrome (SFTS) virus gene according to the present invention and the SFTS diagnostic method using the same can specifically detect the S segment gene of the SFTS virus, and can be performed using already known detection methods. Not only is the accuracy and sensitivity superior to that of the method, but also the SFTS virus can be detected in a short time, so it is effective in quickly and accurately diagnosing severe fever-thrombocytopenia syndrome.

This figure shows the results of rod score measurement after real-time RT-PCR was performed to confirm the SFTS virus detection limit using the SFTS virus gene detection primers and probes devised in the present invention. This figure shows the results of SFTS detection sensitivity and specificity analysis using the SFTS virus gene detection primers and probes of the present invention for specimens obtained on the day of hospitalization of patients suspected of having SFTS in one embodiment of the present invention. This is an example of the present invention, which shows the results of SFTS detection sensitivity and specificity analysis using the SFTS virus gene detection primers and probes of the present invention on specimens obtained from patients suspected of having SFTS during their hospitalization period. be. 1 is a list of bacteria and viruses used as a negative control group for SFTS detection specificity analysis according to the method of the present invention in one embodiment of the present invention. The phylogenetic tree of SFTSV was constructed based on the M segment base sequence of SFTS virus of each patient sample analyzed in one example of the present invention. ~J3) and China clade (C1~C4). In experiments using ticks, 6A is the result of confirming that SFTSV can be detected by real-time RT-PCR analysis using the primers and probe of the present invention on tick samples, and 6B is the result of RT-PCR. This shows the analysis results.

The present invention is characterized in that it provides a new detection composition that can rapidly and accurately detect Severe Fever with Thrombocytopenia Syndrome (SFTS) virus genes.

The present inventors were researching a method capable of detecting SFTS virus-specific genes for accurate and rapid diagnosis and treatment of SFTS infection, and discovered that the S segment of SFTS virus (Small segment; We have discovered primers and probes that can specifically bind to the SFTS virus segment and detect the SFTS virus with high sensitivity and accuracy.

On the other hand, as previously described in the prior art, the Severe Fever with Thrombocytopenia Syndrome (SFTS) virus is a single-stranded RNA virus that belongs to the Bunyaviridae and Phlebovirus genera, and the virus genome consists of three segments. (segment). The three segments of the SFTS virus are the L segment (L segment), which encodes RNA-dependent RNA polymerase, RdRp, and the M segment (Medium segment, which encodes glycoproteins (Gn and Gc), which are constituent components of viral membrane proteins. t , M segment), nucleocapsid protein (NP), which is a viral nucleoprotein and is highly expressed among SFTS virus-derived proteins, induces a cellular immune response in the infected host, and nonstructural S protein (NSs), which has an immune evasion function. It is composed of a small segment (S segment) that codes. According to reports, nonstructural S proteins (NSs) within the S segment of Bunyaviruses enable the virus to counter humans' innate antiviral defenses, making NS proteins one of the major virulence determinants in viral infections. It is well known as one.

In the present invention, it is possible to specifically detect the nonstructural S protein (NSs) gene within the S segment (S segment) among the three segments of the SFTS virus gene isolated from Korean patients and vectors. We conducted experiments to discover primers and probes that could be used, and among the many primers and probes each composed of different base sequences, we discovered primers and probes that had extremely high sensitivity and specificity for the SFTS virus gene. The discovered primers were named SFTS-S 583F primer (5-AGAGGMATGAGCTTGAAC-3; SEQ ID NO: 2) and SFTS-S 653R primer (5-TCCCAACAGTCTAAYAGA-3; SEQ ID NO: 3), and the above probe was SFTS-S 602P. The probe was named (5-ACCACTTATTCACTTCTTCCTCATTGC-3; SEQ ID NO: 4).

Here, the above M may be an A or C base, and the above Y may be a C or T base.

Therefore, the present invention provides a severe fever with thrombocytopenia syndrome (SFTS) virus comprising a pair of primers consisting of the base sequences of SEQ ID NO: 2 and SEQ ID NO: 3; and a probe consisting of the base sequence of SEQ ID NO: 4; The present invention is characterized in that it provides a composition for gene detection.

Further, in the present invention, the primers and probes are derived from the nonstructural S protein (NSs) gene of the S segment of the Severe Fever with Thrombocytopenia Syndrome virus. to specifically bind to The above S segment has a base sequence consisting of SEQ ID NO:1.

Furthermore, in Example 1 of the present invention, new primers and probes were designed for SFTS virus gene detection or diagnosis. In Example 2, real-time RT-PCR was performed using the primers and probes designed for SFTS virus gene detection or diagnosis in Example 1, and the optimal primers and probes were selected. As a result of performing real-time RT-PCR using a pair of primers with SEQ ID NO: 2 and SEQ ID NO: 3 and the probe with SEQ ID NO: 4 discovered in the present invention and analyzing the detection ability of SFTS virus, it was found that primers composed of other base sequences When the primers and probes of the present invention are used, the sensitivity and specificity are the most excellent compared to the conventionally used SFTS virus detection method. As a result, it was found that the present invention can detect SFTS virus genes with higher accuracy and sensitivity than conventional methods.

In Example 2 of the present invention, SFTS virus consisting of the nucleotide sequence of SEQ ID NO: 5 having a size of 71 bp was detected through real-time RT-PCR using a pair of primers of SEQ ID NO: 2 and SEQ ID NO: 3 and a probe of SEQ ID NO: 4. The amplification product of the S segment can be confirmed, and specifically, the base sequence of 71 bp is agaggaatgagcttgaaccaccacttattcacttcttcctcattgcgtaagcctctattagactgttggga (SEQ ID NO: 5).

Further, the composition for detecting the SFTS virus gene according to the present invention is a composition containing the primer and probe of the present invention that can specifically detect the S segment of the SFTS virus with high sensitivity and accuracy, and is a composition that can be used for analysis. It can be used not only for analysis to determine the presence or absence of SFTS virus in a sample, but also as a diagnostic composition for diagnosing Severe Fever with Thrombocytopenia Syndrome (SFTS).

Therefore, the present invention provides a composition for diagnosing Severe Fever with Thrombocytopenia Syndrome (SFTS), which includes a pair of primers of SEQ ID NO: 2 and SEQ ID NO: 3; and a probe of SEQ ID NO: 4; can.

In addition to the above-mentioned pair of primers and the probe according to the present invention, the detection or diagnostic composition of the present invention includes components that can be used in polymerase chain reaction (PCR), such as a reaction buffer solution, dNTP, Mg2+ ion, It can include a DNA polymerase.

The reaction buffer solution can be 1-10mM TrisHCl, 10-40mM KCl (pH 9.0), and the dNTP is one or more selected from dATP, dTTP, dGTP, and dCTP. can be used.

Further, the composition may contain a stabilizer and/or a non-reactive dye for experimental convenience, stabilization, and improvement of reactivity.

The non-reactive dye substance must be selected from substances that do not affect the polymerase chain reaction, and is intended to be used for analysis or identification using polymerase chain reaction products. As substances that meet these conditions, water-soluble dyes such as rhodamine, TAMRA, Lax, bromophenol blue, xylene cyanol, bromocresol red, and cresol red can be used.

The above composition can be provided in a liquid form, and is preferably in a dry state to improve stability, ease of storage, and long-term storage. The above-mentioned drying can be carried out by a known drying method such as normal temperature drying, heating drying, freeze drying, and vacuum drying, and any drying method can be used as long as the components of the composition are not lost. It is possible. The drying method described above can be applied differently depending on the type and amount of enzyme used.

Since the above composition is produced and used in a stabilized state by mixing in one reaction tube and then freezing or drying, a separate mixing process is not required during the polymerase chain reaction. Errors caused by mixing can be prevented, and stability, reactivity and storage properties can be improved.

The above composition may be a commercially available product that already contains the remaining components, except for a pair of primers and a probe, such as a reaction buffer solution, dNTPs, Mg2+ ions, and DNA polymerase.

Furthermore, the present invention can provide a kit for diagnosing severe febrile thrombocytopenia syndrome (SFTS), which includes the composition of the present invention.

The diagnostic kit according to the present invention includes a pair of primers of SEQ ID NO: 2 and SEQ ID NO: 3; and a probe of SEQ ID NO: 4; and is capable of diagnosing Severe Fever Thrombocytopenia Syndrome (SFTS) with very high accuracy. can.

The above diagnosis can use polymerase chain reaction (PCR) to determine whether or not the specimen sample to be analyzed contains the SFTS virus. Reverse transcription polymerase chain reaction (RT-PCR) and real-time polymerase chain reaction (real-time polymerase chain reaction) -time PCR).

In addition to the pair of primers and the probe of the present invention, the diagnostic kit of the present invention also includes reverse transcriptase, polymerase, complementary DNA (cDNA) as template DNA for polymerase chain reaction, and reaction reagents such as buffers. may further include.

The diagnostic kit of the present invention can also include a manual that instructs how to use the kit.

According to one embodiment of the present invention, the diagnostic kit of the present invention can be used for general polymerase chain reaction or real-time polymerase chain reaction, and in this case, reverse transcriptase (reverse transcriptase) is extracted from the RNA genome of SFTS virus. Complementary DNA (cDNA) can be synthesized by separately performing a reverse transcription reaction through the action of transcriptase, and can be used as a template DNA for polymerase chain reaction.

The resultant product of the polymerase chain reaction can be separated by a method such as agarose gel electrophoresis or capillary electrophoresis, and the length corresponding to the DNA polymerized by the pair of primers used can be separated. Severe febrile thrombocytopenia syndrome can be diagnosed by confirming the presence of DNA having the following.

In addition, in the present invention, a probe with SEQ ID NO: 4 that can specifically bind to the SFTS virus gene was used, but the probe was used with a fluorescent substance and a quencher attached to each end. can do. Specifically, a fluorescent substance may be bound to the 5' end of the probe, a quencher may be bound to the 3' end, and a specific wavelength such as red, green, or blue may be bound to the 5' end of the probe. A fluorescent substance that emits ions may be bonded to the 3' end, and a black hole quencher (BHQ) may be bonded to the 3' end. When a fluorescent substance and a quencher are bound to a probe, no fluorescent signal is detected because the quencher absorbs the light emitted by the fluorescent substance. On the other hand, if the fluorescent substance bound by the exonuclease activity of DNA polymerase is separated during the DNA synthesis and elongation process, a fluorescent signal can be detected. While the polymerase chain reaction is progressing, such fluorescent signals can be measured in real time, and the presence or absence of viral genes can be determined based on the increased fluorescent signal. The use of such probes can be usefully used in real-time polymerase chain reactions.

Further, the present invention can provide a method for diagnosing severe fever thrombocytopenia syndrome (SFTS) infection, and can provide a method for providing information for diagnosing SFTS infection.

Preferably, the method for providing information for diagnosing Severe Fever Thrombocytopenia Syndrome (SFTS) infection includes the steps of: (1) isolating viral RNA from a sample isolated from a patient suspected of having Severe Fever Thrombocytopenia Syndrome infection; (2) a step of synthesizing cDNA from the above RNA; (3) a step of performing real-time RT-PCR amplification using the kit used in Example 3 using the cDNA synthesized in step (2) above as a template; and (4) Confirming the gene of severe fever thrombocytopenia syndrome virus through step (3) above;

The sample may be, but is not limited to, blood, tissue, cells, serum, plasma, saliva, sputum, or urine obtained from a mammal; or a tick;

In addition, the severe fever thrombocytopenia syndrome virus gene can be confirmed by any one or more methods selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, and phosphorescence measurement. It can be carried out through.

Furthermore, in the method of the present invention, the above-mentioned SFTS refers to a disease induced by infection with SFTS virus.

As described above, when using the pair of primers of SEQ ID NO: 2 and 3 and the probe of SEQ ID NO: 4 that specifically bind to the S segment of SFTS virus devised in the present invention, SFTS virus can be rapidly detected with high sensitivity and accuracy. As a result of carrying out a real-time RT-PCR analysis method using the primers and probe of the present invention on samples of patients suspected of having SFTS through the following examples of the present invention, it was found that Analysis of patient samples not only confirmed that rapid diagnosis was possible with high sensitivity of 97% and specificity of 100%, but also predicted the sensitivity when hospital admission of SFTS patients was delayed. It was also confirmed that highly accurate diagnosis was possible with a high sensitivity of 88% and a specificity of 100% in an analysis of blood samples collected during the entire hospitalization period of patients suspected of having SFTS.

Furthermore, it has been found that the above-mentioned diagnostic accuracy achieved by the method of the present invention is significantly superior to that achieved by conventionally used diagnostic methods and when other types of primers and probes are used. Ta.

Therefore, the method for diagnosing SFTS infection and the method for providing information for diagnosing SFTS using the composition for detecting SFTS devised in the present invention can diagnose SFTS quickly and accurately at an early stage, and can detect SFTS virus in real time. As a result, it is possible to diagnose with high accuracy even after a considerable period of time has passed since the onset of the disease, and to distinguish SFTS from other diseases with high specificity.

Hereinafter, the present invention will be explained in more detail through Examples. These Examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these Examples.

<Example 1>
Primer and probe production for SFTS virus gene detection or diagnosis

The present inventors have designed new diagnostic primers and probes targeting the S segment sequence of SFTS virus that can rapidly, accurately, and highly sensitively detect severe fever thrombocytopenia syndrome as shown in Table 2 below. .

In addition, for designing the primers and probes shown in Table 2 below, the S segment sequences of SFTS virus listed in Tables 1A to 1C below were used as reference.

Tables 1A to 1C: Information on the S segment of SFTS viruses identified from patients in South Korea, China, and Japan

Table 2: Primers and probes designed for SFTS virus gene detection

In the table above, M may be an A or C base, and Y may be a C or T base.

<Example 2>
Optimal primer and probe selection for SFTS virus gene detection or diagnosis

Real-time RT-PCR was performed using the primers and probes designed in Example 1 for detecting or diagnosing the SFTS virus gene, and sensitivity and specificity tests were performed.

As a result, as shown in Figure 1, when the limit of detection (LOD) was confirmed using plasmid DNA (recombinant vector DNA in which the S segment of SFTS virus was cloned), the primers and the LOD in Table 2 above were confirmed. Among the probes, No. The use of primers and probe No. 1 showed superior sensitivity and specificity compared to groups using other primers and probes, and was also superior to existing test methods for SFTS virus detection. The sensitivity and specificity were demonstrated. Specifically, No. When using primers and probe 1, real-time RT-PCR analysis confirmed that 95% detection (LOD 95%) was 39.739 copies/test (ct 40.3), and the existing SFTS virus detection test method was performed. Sometimes 96.332 copies/test (ct 38.9), and due to the higher sensitivity of real-time RT-PCR when using the primers and probes devised in the present invention, it is recommended for the detection and diagnosis of SFTS virus genes. It was found that both time and cost can be saved and the detection effect is excellent.

<Example 3>
SFTS virus gene detection or SFTS detectability analysis using diagnostic primers and probes of the present invention in samples obtained from patients

The present inventors have proposed primers and probes for SFTS virus gene detection or diagnosis devised in the present invention (primers No. 1 in Table 1 above (SFTS-S 583F, 653R primers) and probes (SFTS-S 602P probe)). In order to confirm whether it can actually be used to diagnose SFTS infection, the following experiment was performed.

For this purpose, the samples used in the experiment were obtained from patients satisfying the following three conditions and analyzed.

1. Those who are 19 years of age or older 2. Persons considered to be infected with SFTS Persons for whom SFTS symptoms have been objectively confirmed from the onset of symptoms to the time of study participation evaluation, and those who fall under the following - Fever (axillary temperature >37.8℃)
- Thrombocytopenia (platelet<100 X 109/L)
-Leukopenia (WBC<5 x 109/L)
3. When SFTS is suspected by an infectious physician

SFTS RT-PCR tests were performed on patients who met the above three conditions, and a definitive diagnosis of SFTS was defined as a 4-fold increase in IFA or virus isolation, and each PCR method was performed to evaluate the accuracy.

In addition, sensitivity confirmation samples were collected and used for SFTS patients in 2018, and 119 blood samples were collected from 36 patients, and samples for specificity confirmation were collected between 2008 and 2018. The study was conducted on 50 patients who were confirmed to have other diseases other than SFTS using sample samples. For reference, a list of pathogens and viruses used as a negative control group for specificity testing is shown in Figure 4.

Other PCR was performed to compare sensitivity and specificity, but viral RNA was extracted from plasma and WB (whole blood) of patients, and the above extraction was performed using Virus Gene-spinTM Viral DNA/RNA Extraction Kit (intron). It was carried out using Thereafter, cDNA was synthesized from the above RNA using SuperScript VILO MasterMix (Invitrogen) and used as template DNA, and real-time RT-PCR was performed as follows.

Real-time RT-PCR targeting the S segment of the SFTS virus is performed by the method using the primers and probes devised in the present invention and the previously disclosed method of PCR shown in Table 3 below, and targets the S segment. Nested RT-PCR and RT-PCR targeting the M segment were performed. Real-time RT-PCR was performed on a 96 Excycler (Bioneer) instrument using a LightCycler TaqMan Master (Roche Diagnostics, Indianapolis). RT-PCR and nested RT-PCR were performed using Accupower PCR premix (Bioneer) and cDNA. Information on each PCR performed is shown in Table 4 below.

Table 3: Primer and probe information used for PCR

Table 4: PCR performance conditions

Table 5: Sensitivity and specificity results

As a result of the analysis, as shown in Table 5 above, the real-time RT-PCR analysis results performed using the primers and probes devised in the present invention had the best values in terms of sensitivity and specificity compared to other PCR methods. It was found that the detection effect was the best, and it was shown that the SFTS virus could be accurately diagnosed and detected in a short time of 1 to 2 hours compared to other existing methods.

<Example 4>
Verification of SFTS detection accuracy using SFTS virus gene detection or diagnostic primers and probes of the present invention

<4-1> Accuracy of PCR diagnostic method using specimens obtained at the time of hospital visit SFTS was detected using the PCR analysis method using primers and probes for detecting the SFTS virus gene devised in the present invention using patient samples (blood) at the time of hospital visit. Sensitivity and specificity validation analysis for detection was performed.

Table 6: Accuracy of PCR diagnostic method using samples at the time of visit

-SQ ref, real-time RT-PCR (S-segment, reference); SQ design, real-time RT-PCR (S-segment, design, present invention)

Table 7: Comparison of accuracy of PCR diagnostic method using samples at the time of visit

-SQ ref, real-time RT-PCR (S-segment, reference); SQ design, real-time RT-PCR (S-segment, design, claimed invention)

As a result, as shown in Table 6 and Figure 2 above, as a result of real-time RT-PCR analysis targeting the S segment of SFTS, the group using the primers and probe devised in the present invention was found to be superior to the group using other types of primers and probes. It showed the highest sensitivity and specificity compared to the group using a probe, and specifically, it was possible to confirm the results of a sensitivity of 97.2% and a specificity of 100%. On the other hand, nested RT-PCR analysis targeting the S segment showed a sensitivity of 91.67%, but nested RT-PCR requires the hassle of performing two PCRs, which can cause PCR contamination. It was found that using the primers and probes of the present invention can lead to more accurate and better test results when considering the higher sensitivity.

In addition, with respect to the above results, a comparative analysis of each sensitivity and specificity using AUC revealed that the real-time RT-PCR method using the primers and probe devised in the present invention is a conventional RT-PCR method targeting the M segment. - It was confirmed that the accuracy was statistically significantly higher than that of PCR (see Table 7).

<4-2> Accuracy evaluation of the PCR diagnostic method using SFTS virus gene detection or diagnostic primers and probes of the present invention using blood samples collected during the hospitalization period of patients suspected of SFTS

Further, the present inventors performed a sensitivity and specificity analysis for SFTS detection using the primers and probes devised in the present invention on blood samples obtained from patients suspected of having SFTS during their hospitalization period. Specifically, a confirmed diagnosis of SFTS is defined as a 4-fold increase in IFA or virus isolation and confirmation, and 119 blood samples were collected from 36 patients who were confirmed to be SFTS patients and 50 patients who were confirmed to have other diseases. An accuracy test was performed using the method described in Example <4-1> above.

In addition, the analysis was carried out on blood samples taken during the hospitalization period of patients suspected of having SFTS because patients usually do not come to the hospital early even though they develop symptoms, but often come late, and this may be due to other diseases. There are many cases where SFTS is suspected during treatment and a request is made for SFTS PCR testing during the hospitalization period. This is to confirm that it is possible to diagnose SFTS with high sensitivity and specificity even when using samples from patients who came to the hospital mid or late.

Table 8: Accuracy of PCR diagnostic method using specimens during hospitalization period

-SQ ref, real-time RT-PCR (S-segment, reference); SQ design, real-time RT-PCR (S-segment, design, present invention)

Table 9: Comparison of accuracy of PCR diagnostic method using specimens during hospitalization period

-SQ ref, real-time RT-PCR (S-segment, reference); SQ design, real-time RT-PCR (S-segment, design, present invention)

As a result, as shown in Tables 8 to 9 and FIG. It has been shown to be statistically significantly more accurate than time RT-PCR and nested RT-PCR, and has superior sensitivity compared to other primers and probes. It shows.

<4-3> Accuracy analysis of PCR diagnostic method using SFTS virus gene detection or diagnostic primers and probes of the present invention for various pathogens and viruses

Specificity was analyzed using the primers and probes devised in the present invention for the bacteria and virus shown in FIG.

As a result, it was confirmed that all bacteria that cause various diseases were negative, and when other viruses other than SFTSV were detected, they were not detected, including dengue virus, influenza virus, hepatitis A virus, and Hantan virus. It was confirmed that none of the above was detected in samples from patients infected with (Hantaan virus).

Therefore, these results show that the primers and probes devised in the present invention exhibit excellent specificity for the SFTS virus, and no amplification products were detected for other bacterial strains or viruses. It was found that the SFTS virus could be specifically detected.

<Example 5>
SFTS virus detectability analysis of Korean, Japanese and Chinese phylogenetic groups

According to recent research results, it has been confirmed that the genetic sequences of SFTS viruses, which cause severe fever and thrombocytopenia syndrome that occur in different countries, are different from each other. On the other hand, although a detection technology that can simultaneously detect SFTS viruses of various clades has not yet been developed, the present inventors believe that when using the primers and probes devised in the present invention, In order to confirm whether all SFTS viruses in the group could be detected, the following experiment was performed.

Specifically, SFTS virus is classified into Korea, Japan, and China clades when analyzed by phylogenetic group based on the virus gene nucleotide sequence, and SFTS patients in South Korea have been confirmed to belong to all three clades. Therefore, as shown in Tables 10A and 10B below, real-time RT-PCR was performed using the primers and probes of the present invention on samples from patients infected with SFTSV of different phylogenetic groups as in the above-described embodiments. Ta. Furthermore, the phylogenetic group of SFTSV used in the present invention and the classification for each sample are shown in FIG.

Table 10A-Table 10B: SFTS virus detectability analysis of Korean, Japanese and Chinese phylogenetic groups

-C, Chinese clade; J, Japanese clade; K, Korean clade; SQ design, real-time RT-PCR (S-segment, design, present invention)

As a result of the analysis, as shown in Table 10 above, when using the primers and probes of the present invention in samples from all SFTS patients infected with clade from three countries, all the amplified products could be confirmed. Through these results, the present inventors found that the method devised in the present invention can detect SFTS viruses of Korean, Japanese, and Chinese phylogenetic groups.

<Example 6>
SFTS virus gene detection of the present invention or detection of SFTS using diagnostic primers and probes in tick samples

55 ticks were collected from a patient who came to the hospital after being bitten by a tick, and RT-PCR for SFTSV was performed on these using the primers (SEQ ID NO: 2, 3) and probe (SEQ ID NO: 4) of the present invention. As a result, 54 tick samples were confirmed to be negative, and 1 tick sample was confirmed to be positive. Similarly, in the nested RT-PCR test, 54 tick samples were confirmed to be negative, and only one tick was confirmed to be positive, and the results of the real-time RT-PCR of the present invention were also confirmed to be positive. As a result of nucleotide sequence analysis, it was confirmed to be SFTS virus (see Figure 6).

Therefore, through these results, the present inventors found that the primers and probes devised in the present invention are capable of detecting SFTSV not only in human-derived samples but also in tick samples (SFTS SQ-PCR). (ref) Ct value of 30.25 and SFTS SQ-PCR (design) Ct value of 27.98) (see Figure 6), these results are consistent with the RT-PCR using the primers and probes of the present invention. It was found that SFTSV could also be detected through PCR analysis results (see Figure 6).

Through the above results, the present inventors have devised the present invention that can detect and amplify the base sequence of the nonstructural S protein (NSs) gene within the S segment of the SFTS virus, which is the causative virus of severe fever and thrombocytopenia syndrome. When real-time RT-PCR is performed using the primers and probes developed, severe fever and thrombocytopenia syndrome caused by the genetic type of virus prevalent not only in Korea but also in China and Japan can be detected accurately and quickly, and has excellent accuracy. It turns out that it can be diagnosed based on the degree of

The present invention has been discussed above, focusing on its preferred embodiments. Those skilled in the art to which the present invention pertains should be able to understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the present invention. Accordingly, the disclosed embodiments are to be considered in an illustrative rather than a restrictive light. The scope of the present invention is indicated in the claims rather than the above description, and it can be said that all differences within the equivalent range must be construed as being included in the present invention.

Claims (7)

A composition for detecting a severe fever with thrombocytopenia syndrome (SFTS) virus gene, comprising a pair of primers of SEQ ID NO: 2 and SEQ ID NO: 3; and a probe of SEQ ID NO: 4. The pair of primers of SEQ ID NO: 2 and SEQ ID NO: 3; and the probe of SEQ ID NO: 4; The composition for detecting the severe fever thrombocytopenia syndrome (SFTS) virus gene according to claim 1, wherein the composition specifically binds to the virus. A diagnostic kit for Severe Fever Thrombocytopenia Syndrome (SFTS), comprising the composition of claim 1. (1) Isolating viral RNA from a sample isolated from a patient suspected of having severe fever and thrombocytopenia syndrome;
(2) step of synthesizing cDNA from the above RNA;
(3) using the cDNA synthesized in step (2) as a template and performing real-time RT-PCR amplification using the kit of claim 3; and (4) performing severe fever thrombocytopenia syndrome virus through step (3) above. confirming the gene of;
A method for providing information for diagnosing Severe Fever Thrombocytopenia Syndrome (SFTS) infection. Severely febrile platelets according to claim 4, wherein the sample in step (1) is blood, tissue, cells, serum, plasma, saliva, sputum, or urine obtained from a mammal; or a tick; Information provision method for diagnosis of infection with reduced syndrome syndrome (SFTS). Confirmation of the severe fever thrombocytopenia syndrome virus gene in step (3) above is performed using one or more methods selected from the group consisting of capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, and phosphorescence measurement. The method for providing information for diagnosing severe fever thrombocytopenia syndrome (SFTS) infection according to claim 4, characterized in that the method is carried out by the method. 5. The gene of the severe fever thrombocytopenia syndrome virus in step (3) is the S segment (S segment) of the severe fever thrombocytopenia syndrome virus consisting of SEQ ID NO: 1. A method for providing information for diagnosing Severe Fever Thrombocytopenia Syndrome (SFTS) infection.